This work will examine the factors which govern the rate of transition between equilibrium states of an allosteric protein. It is an attempt to test models for the equilibrium states, and to determine if the factors which are significant in determining the free energy of the equilibrium states are equally significant in determining the rates of transition between those states. Hemoglobin has been selected as a model allosteric protein because it has already been extensively described in structural and thermodynamic terms. The forward and reverse rates of transition between the T and R conformations of hemoglobin are to be measured using the method of modulated excitation spectroscopy. Conformational change will be monitored by optical probes sensitive to the environment of the heme and to the environment of the subunit interfaces, and by the use of a DPG analog whose fluorescence is quenched upon binding between the beta subunits (8-hydroxy- 1,2,6 pyrene trisulfonate, or PTS) Measurements will be performed as a function of pH and temperature. In addition to studies with carbon monoxide as a ligand, we will also gather data for conformational change rates with oxygen bound. By a novel application of the modulation technique we will measure the rates of binding and release of oxygen in the T and R states. This will provide an independent measure of the allosteric constant c. These measurements will allow a precise comparison to be made between predicted differences in T and R with three ligands bound, and measured differences obtained by modulation. The use of cobalt-iron hybrids will reveal how metal substitution affects the quaternary structure change kinetics. With oxygen as a ligand, the rate of allosteric change can be studied for unligated alpha or beta chains. Similarly, the rate of ligand binding to alpha or beta chains can be determined. With CO as the ligand, the rate of allosteric change with two ligands bound can be measured. In a novel approach to modulation, the kinetics of fully cobalt substituted hemoglobin will be studied by modulating the uptake of solution oxygen by photolysis of an oxygen scavenger such as myoglobin. Through the use of indicator dyes the synchrony of proton release with ligand binding at the last step of ligation can also be studied. Finally, equilibrium measurements of oxygen binding will provide allosteric reference spectra as well as allosteric parameters for the specific conditions employed (buffers, pH, temperature). These experiments will further serve to characterize the transition state between the alternative hemoglobin structures, and relate it to molecular descriptions of the allosteric change. The work done here generates rate pairs, so that equilibrium data about relative stabilities of structures is a natural byproduct. This equilibrium information will be used to test thermodynamic theories describing hemoglobin function.